Multiplex detection of hepatitis virus variations

ABSTRACT

Disclosed is a method for the detection of virus variations. Disclosed also is a method for the treatment of virus infection in a subject based on the detection of virus variations. Additionally, a kit is provided for the detection of virus variations.

This claims priority from U.S. Provisional Application Ser. No. 61/175,827 filed May 5, 2009. The entirety of that provisional application is incorporated herein by reference.

FIELD

The present invention generally relates to a method and a kit for the detection of virus variations.

BACKGROUND

Hepatitis is an inflammation of the liver, most commonly caused by a viral infection. There are five main hepatitis viruses, referred to as types A, B, C, D and E.

Hepatitis A and E are typically caused by ingestion of contaminated food or water. Hepatitis B, C and D usually occur as a result of parenteral contact with infected body fluids (e.g. from blood transfusions or invasive medical procedures using contaminated equipment). Hepatitis B is also transmitted by sexual contact.

Worldwide, it is estimated that 400 million people are chronically infected with hepatitis B virus (HBV). Chronic hepatitis B (CHB) infection is the most common cause of liver cirrhosis and hepatocellular carcinoma (HCC), with an estimated 500,000-900,000 death per year. Continuing HBV replication increases the risk of progression to cirrhosis and HCC (28-31).

Variations in the hepatitis B virus (HBV) genome may develop spontaneously or under selective pressure from antiviral therapy. Some of these variations confer drug resistance, resulting in treatment failure which may further lead to hepatitis reactivation and hepatic decompensation (32).

SUMMARY

One aspect is to provide a method for the detection of hepatitis virus (HV) variations, comprising a) providing a DNA molecule of an HV from a subject suffering from the HV infection as a template, wherein the DNA molecule includes an HV variation site; b) extending multiplex extension primers along the template to obtain extension products containing nucleotides at the variation sites; and c) analyzing the extension products by mass spectrometry (MS) to detect the HV variations.

Another aspect is to provide a method for the treatment of a hepatitis virus (HV) infection in a subject, comprising a) detecting HV variations in the subject according to the detection method disclosed herein, where the HV variations are in association with drug resistance; b) assessing the drug resistance of the subject based on the detection; and c) treating the subject based on the assessment.

Yet another aspect is to provide a kit for the detection of hepatitis virus (HV) variations by mass spectrometry analysis, comprising multiplex extension primers disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representative mass spectrum for multiplex extension assay 1. The entire raw spectrum is shown in panel a. The intensities for the peaks are arbitrary units. Panel b zooms in one assay (nt_(—)367). “Primer” represents the unextended primer. T and C represent the extension products for the mutant sequence (T) and wild type sequence (C), respectively.

FIG. 2 is an illustration showing call rates for each variation and call rates for each sample. In panel a, call rates for each variation for each group (TN for treatment naive and DR for drug resistant) are shown. Majority of the variations had higher call rates (88.3% of the variations achieved a call rate over 90%). In panel b, call rates for each sample (shown by its viral load on the X axis) are shown. Drug resistant samples were more likely to have lower call rates. All samples with call rates <90% were drug resistant samples.

FIG. 3 is an extension reaction thermal profile. A 200-short-cycle program with two cycling loops was used for extension reaction. The 5-cycle loop sat inside the 40-cycle loop.

FIG. 4 depicts alignment of PCR primers to different HBV genotypes. Four primers were designed, 251F and 1004R for the reverse transcriptase, and 1593F and 1950R for precore and core promoters.

FIG. 5 is an exemplified mass spectrum. Panel a) MS detected both wild type and mutant sequences while sequencing only detected one sequence; panel b) sequencing detected both wild type and mutant sequences while MS only detected one sequence.

FIG. 6 shows examples of extension primers. MW stands for molecular weight, UEP for an unextended primer, and EP for an extended primer.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwise indicated, conventional methods of molecular biology, virology, cell biology, microbiology and biochemistry within the skill of the art. Such techniques are explained fully in the literature. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example to understand the document where the term is originally used).

As used herein, the teen “variation” refers to the genetic variation of a virus.

As used herein, the term “variation site” is intended to mean the nucleotide site where a variation is present.

As used herein, the term “DNA” is intended to mean any polymer of deoxynucleotides.

As used herein, the term “complementary” or “complementarity” is used in reference to nucleotide/polynucleotides related by the base-pairing rules. For polynucleotides, “complementary” may be “partial,” in which only some of the polynucleotides' bases are matched according to the base-pairing rules. Or, there may be “complete” or “total” complementarity between the polynucleotides.

One aspect disclosed herein is directed to a method for the detection of hepatitis virus (HV) variations, comprising the steps of a) providing a DNA molecule of an HV from a subject suffering from the HV infection as a template, wherein the DNA molecule includes an HV variation site; b) extending multiplex extension primers along the template to obtain extension products containing nucleotides at the variation sites; and c) analyzing the extension products by mass spectrometry (MS) to detect the HV variations.

Exemplary HVs are hepatitis A, B, C and D viruses. Preferably, the HV is an HBV.

HV variations occur at much higher density on an HV genome as compared with human SNPs. HV may exist as quasi-species such that some HV variations may only be present at a minor proportion, while heterozygous SNPs are present at 1:1 ratio. The HV variations include, but not limited to, variations in association with virulence, viral escape and/or drug resistance of an HV. In some embodiments, the HV variations are variations in association with drug resistance of an HV, including HV variations that have been documented for their functional roles in drug resistance and/or HV variations that have been detected in serum samples derived from patients undergoing an anti-HV therapy. In a particular embodiment, where the HV is an HBV, the variations which are associated with drug resistance are exemplarily listed in Table 1.

TABLE 1 Summary of mutations Amino acid HBV Region substitutions Nucleotide changes Reference Reverse rtT70S A/T@337 (1) transcriptase rtN71T A/C@341 (1) rtS78T T/A@361 (2) rtL80I(V) C/T/A/G@367 (3) rtL82M C/A@373 (2, 4) rtV84M G/A@379 (5) rtS85A T/G@382 (5) rtA86P G/C@385 (1) rtP92L C/T@404 (1) rtT128N C/A@512 (6) rtH133L A/T@527 (7) rtS/T135C A/T@532 (4) C/G@533 rtI/V163V A/G@616 (2) rtF166L T/C@ 625 (8) rtI169T T/C@635 (9) rtV173L G/C/T@646 (10)  rtP177L C/T@659 (2) rtL179P T/C@665 (11)  rtL180M C/T/A@667 (12)  rtA181T(V/S) G/A/T@670 (13)  C/T@671 rtT184G A/G@679 (9) C/G@680 rtV191I G/A@700 (1) rtA194T G/A@709 (14)  rtA200V C/T@728 (2) rtS202I G/T@734 (9) rtM204V/I A/G@739 (4) G/C/T@741 rtM204S T/G@740 (15)  G/T@741 rtV207I G/A@748 (16)  G/A/T/C@750 rtS213T T/A@766 (11, 17) rtV214A T/C@770 (5) Reverse rtQ215S C/T@772 (18)  transcriptase rtS219T T/A@784 (6) rtF221Y T/A@791 (19)  rtS223A T/G@796 (20, 21) rtI224V A/G@799 (22)  rtL229V/M T/G/A@814  (4, 23) rtI233V A/G@826 (24)  rtH234Q T/A/G@831 (7) rtL235I T/C/A@832 (2) rtN236T A/C@836 (25)  rtP237H C/A@839 (5) rtN/S/H/A238S A/C/G@841  (2, 5), T/C@843 rtY245S A/C@863 (17)  rtN/H248H A/C@871 (1, 2) rtM250V A/G@877 (9) rtV/I253I G/A@886 (1, 2) Precore/Core G1896A (26)  C1858T (27)  Basal core A1762T (27)  promoter G1764A (27) 

The DNA molecules of an HV disclosed herein include, for example but not limited to, the coding sequences, the non-coding sequences of an HV, and fragments thereof. In some embodiments, the DNA molecule is a HBV reverse transcriptase gene, a HBV basal core/precore promoter, or a fragment thereof.

One or more different DNA molecules can be provided, where each DNA molecule includes at least one HV variation site of interest. In a particular embodiment, the HV variation sites are the HBV variation sites as listed in Table 1.

The DNA molecule can be provided from a sample of a subject suffering from an HV infection. The subject can either be the one who is treatment naive, or the one who is drug resistance. Exemplary samples include, but not limited to, hepatocytes, specimen of liver tissue, serum, plasma and blood of the subject. In some embodiments, the DNA molecule can be provided from samples of more than one subject.

In one embodiment the DNA molecule can be extracted and/or amplified from the sample as disclosed herein.

Extraction of the DNA molecule can be performed using a commercially available DNA preparation kit, in particular, a DNA extraction kit for an animal according to the manufacturer's protocol.

In some embodiments, the HV RNA is extracted from the sample of the subject and then reversed to the DNA molecule as disclosed herein. RNA extraction and reverse reaction can be performed using commercially available kits.

In some embodiments, before extending multiplex extension primers, amplification of the DNA molecule from the extracted DNA or RNA can be performed by means of PCR, RT-PCR, or the like using amplification primers. PCR, RT-PCR or the like can be performed using a commercially available kit according to the manufacturer's protocol. The thermal profile of PCR, RT-PCR or the like, or the optimization thereof can be determined or performed by an artisan given a specific DNA molecule to be amplified and specific amplification primers.

The amplification primers can be designed according to the DNA molecule to be amplified. In a preferred embodiment, the amplification primers can be designed by aligning genomic sequences from different HV genotypes. In another preferred embodiment, sequence tag(s) can be added to 5′-end of the amplification primers to increase their molecular weights for avoidance of any interference in mass spectra. In yet another preferred embodiment, the 5′- and 3′-portions of the DNA molecule correspond to the highly conserved genomic regions of an HV, where each portion represents an amplification primer.

The amplification primers can be 10-50 nucleotides in length, preferably 15-35 in length.

In some embodiments, the amplification can be a multiplex amplification when the HV variation sites are present in more than one DNA molecule. The multiplex can range from 2-plex to 50-plex, in particular, from 2-plex to 30-plex. The amplification primers can be multiplex amplification primers which are designed for different DNA molecules. In a preferred embodiment, the multiplex amplification primers are designed to avoid primer cross-hybridization. In a particular embodiment, the multiplex amplification primers are the primers having a 70%, 80%, 85%, preferably 90%, more preferably 95%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 1-4.

In a particular embodiment, the DNA molecule is amplified by a 2-plex PCR using the primers as set forth in SEQ ID NOs: 1-4. The PCR reactions (25 μL) may contain 5 μL HBV DNA, 200 nM each of the primers (SEQ ID NOs: 1-2), 100 nM each of the primers (SEQ ID NOs: 3-4), 1× buffer (with 1.5 mM Mg2+), 1.0 mM additional Mg2+, 200 μM dNTP (each), and 0.5 units of Hotstar Tag polymerase (Qiagen). PCR may be initiated at 95° C. for 15 min, followed by 45 cycles of denaturation at 95° C. for 40 sec, annealing at 57° C. for 30 sec, and extension at 72° C. for 1.5 mM and a final extension at 72° C. for 3 min.

The DNA molecule may be provided after further treatment. The treatment can be dephosphorylation. In some embodiments, the amplified DNA molecule can be treated with a dephosphorylating reagent including, but not limited to, shrimp alkaline phosphatase (SAP).

In some embodiments, the multiplex extension as disclosed herein can preferably be a single nucleotide extension. The multiplex extension is used to extend the multiple primers to include nucleotides at more than one variation site of an HV. The extension can be in a direction of from 5′ to 3′ of an extension primer. The multiplex can range from 2-plex to 72-plex, in particular, from 2-plex to 36-plex. The extension can be performed using a commercially available primer extension kit. In particular, the primer extension kit is the one contained in MassARRAY iPLEX™ (Sequenom, Inc, San Diego, Calif.) for primer extension, an iPLEX™ reaction. The thermal profile of the extension or the optimization thereof can be determined or performed by an artisan.

In some embodiments, a standard thermal profile for MassARRAY iPLEX™ is employed as listed in FIG. 3. The extension reaction mixture may contain 7 μL of PCR-SAP products, 0.2 μL of 10× iPLEX buffer plus, 0.2 μL iPLEX termination mix with modified ddNTPs, 0.94 μL extension primer mixture, 0.041 μL iPLEX thermosequenase.

The multiplex extension primers are designed for more than one HV variation. Each multiplex extension primer can be designed based on its target HV variation site. In particular, a multiplex extension primer can be designed to be hybridizable to the immediate 3′ downstream to a nucleotide at an HV variation site. More particularly, a multiplex extension primer can be complementary to the immediate 3′ downstream to a nucleotide at an HV variation site. Even more particularly, the 3′-end of a multiplex extension primer can be complementary to the very nucleotide linked to the 3′ of a nucleotide at an HV variation site.

In some embodiments, a multiplex extension primer can be a mixture of degenerate primers directed to the same target variation site where another one or more HV variation sites appear adjacent to the 3′-end of the nucleotide at the target variation site. In a preferred embodiment, a sequence tag can be added to 5′-end of a multiplex extension primer to increase its molecular weight for avoidance of any interference in mass spectra. The multiplex extension primers can be 10-50 nucleotides in length, particularly 15-35 in length, more particularly 15-28 in length.

The concentrations of multiplex extension primers can be adjusted to compensate differences in signal intensities in MS due to differences in sequence and molecular weight among different multiplex extension primers. Adjustments can be made based on real mass spectral data generated.

In one embodiment, the extension is 12-plex using the multiplex extension primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 5-16.

In another embodiment, the extension is 17-plex using the multiplex extension primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 17-33.

In yet another embodiment, the extension is 17-plex using the multiplex extension primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 34-50.

In still yet another embodiment, the extension is 14-plex using the multiplex extension primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 51-64.

The extension products disclosed herein, i.e. the extended multiplex extension primers, can be analyzed by MS, identifying the nucleotides at HV variation sites which are also contained in the extension products. MS analysis can be performed by a commercially available MS device according to the manufacturer's protocol. Data analysis can be performed using commercially available MS data analysis software.

In some embodiments, the MS can be matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

The MS analysis may comprise a step of extension product conditioning such as desaltification prior to sample loading on a MS device.

Another aspect is directed to a method for the treatment of a hepatitis virus (HV) infection in a subject, comprising the steps of a) detecting HV variations in the subject according to the detection method disclosed herein, wherein the HV variations are in association with drug resistance; b) assessing the drug resistance of the subject based on the detection; and c) treating the subject based on the assessment.

Yet another aspect is directed to a kit for the detection of hepatitis virus (HV) variations by mass spectrometry analysis, comprising multiplex extension primers disclosed herein.

In one embodiment, the multiplex extension primers are the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 5-16.

In another embodiment, the multiplex extension primers are the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 17-33.

In yet another embodiment, the multiplex extension primers are the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 34-50.

In still yet another embodiment, the multiplex extension primers are the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 51-64.

The kit may further comprise amplification primers as disclosed herein.

In a particular embodiment, the amplification primers are the primers having a 70%, 80%, 85%, preferably 90%, more preferably 95%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 1-4.

The kit may still further comprise reagents for primer extension.

The kit may still further comprise reagents for primer extension, reagents for dephosphorylation, and/or reagents for PCR. In a particular embodiment, the kit may still further comprise the reagents as contained in MassARRAY iPLEX™.

EXAMPLES Example 1 HBV Variation Selection

A literature search was performed to include all known HBV mutations that are directly associated with drug resistance. Variations in the precore (G1896A and C1858T) and basal core promoter (A1762T and G1764A) were included. Variations frequently observed during antiviral treatment, but not conclusively implicated in drug resistance were also included with the hope that when a large number of samples are analyzed in the future, statistical significance for their association with antiviral treatment may be observed for some of them. Summary of mutations are listed in Table 1.

Example 2 Sample Collection

A total of 168 patients with HBV infection were recruited for this study after obtaining informed consent. Out of these 168 patients, 88 patients had not received any antiviral therapy (treatment naive) and 80 patients had experienced a virological breakthrough with HBV DNA increase by >1 log from nadir level and to >100,000 copies/ml. About 1.5 mL serum samples were collected for each patient.

HBV DNA was extracted from serum using the QIAamp DNA Blood Mini Kit (Qiagen) according to the “DNA Purification from Blood or Body Fluids (Spin Protocol)”. 800 μL serum was used for DNA extraction into 50 μL elution volume. Viral load of each sample was determined using a fluorescence PCR kit (PG Biotech, Shenzhen, China) (12).

HBV DNA ranged from 10^(3.07) to 10^(8.80) copies/mL (median: 10^(7.38)) and 10^(3.42) to 10^(8.21) (median: 10^(5.39)) copies/mL for the 88 treatment naive patients and the 80 drug resistant patients, respectively. The viral load in treatment naive samples was significantly higher than drug resistant samples (p<0.05, t-test).

Moreover, based on capillary sequencing of the samples from 32 treatment naive patients, 23 (71.9%) were genotype B HBV and 9 (28.1%) were genotype C HBV. The HBV DNA from thirty-three drug resistant samples was also sequenced. Twenty three (69.7%) patients were infected by genotype B HBV and 10 (30.3%) by genotype C HBV.

Example 3 HBV Variation Detection

Four main steps were involved in the detection of HBV variations by MALDI-TOF MS. The target HBV variations are listed in Table 1. Sequences and molecular weights of extension primers and extension products are provided in FIG. 6. All primers were synthesized by Integrated DNA Technologies (Coralville, Iowa). All other reagents were purchased from Sequenom (California, USA) unless otherwise specified.

Step 1: 2-Plex PCR to Amplify the HBV Regions of Interest

The reverse transcriptase and the basal core promoter/precore regions were selected since these two regions contain the functionally important variations as well as the variations frequently observed in patients undergoing antiviral therapy. We designed a 2-plex PCR assay which amplified all the target variations.

PCR primers were designed by aligning genomic sequences from different HBV genotypes (FIG. 4). PCR primer pairs were 5′-gttggatgGACTCGTGGTGGACTTCT CTCA-3′ (251F-tag) (SEQ ID NO: 1) and 5′-ggatgCCCACAATTCKTTGACATACTT TCC-3′ (1004R-tag) (SEQ ID NO: 2), and 5′-acgttggatgACCTCTGCACGTYRCATGGA-3′ (1593F-tag) (SEQ ID NO: 3) and 5′-ggatgGAGAGTAACTCCACAGTAGCTCCAA-3′ (1950R-tag) (SEQ ID NO: 4), respectively. The non-capital letters were sequence tags to increase the molecular weights of the PCR primers so that they would not interfere in mass spectra.

The reverse transcriptase and the basal core promoter/precore regions were amplified by a 2-plex PCR using the PCR primer pairs above, generating two amplification products at a length of 754 by and of 358 bp.

The 25 μL PCR reactions contained 5 μL HBV DNA, 200 nM each of the primers (251F-tag and 1004R-tag), 100 nM each of the primers (1593F-tag and 1950R-tag), 1× buffer (with 1.5 mM Mg²⁺), 1.0 mM additional Mg²⁺, 200 μM dNTP (each), and 0.5 units of Hotstar Taq polymerase (Qiagen).

PCR was initiated at 95° C. for 15 min, followed by 45 cycles of denaturation at 95° C. for 40 sec, annealing at 57° C. for 30 see, and extension at 72° C. for 1.5 min and a final extension at 72° C. for 3 min.

Upon further PCR optimization, this 2-plex PCR assay consistently amplified HBV DNA when the input DNA copy number was more than 94 copies per PCR. This allowed us to analyze all serum samples when the viral load was over 1170 copies/mL serum.

Step 2: Shrimp Alkaline Phosphatase (SAP) Treatment

To remove the remaining dNTPs in the PCR reactions, a 2-μL SAP solution including 1.53 μL of H₂O, 0.17 μL of SAP 10× buffer, and 0.5 units of SAP enzyme, was mixed with 5 μL of the PCR products. The reaction was performed at 37° C. for 40 min followed by inactivating at 85° C. for 5 min.

Step 3: Multiplex Primer Extension Reactions

MassARRAY Assay design (Sequenom) can achieve a multiplex level as high as 36. However, we decided to design assays at significantly lower multiplex levels so that new variations could be added with ease in future updates. As a result, the 60 target HBV variations were analyzed in 4 separate primer extension reactions (12-plex in assay 1, 17-plex in assay 2, 17-plex in assay 3, and 14-plex in assay 4).

60 extension primers were required for the 60 variations (FIG. 6).

The 9-μL extension reactions contained 7 μL of amplification-dephosphorylation products, 0.2 μL of 10× iPLEX buffer plus, 0.2 μL iPLEX termination mix with modified ddNTPs, 0.94 μL extension primer mixture, 0.041 μL iPLEX thermosequenase. A standard thermal profile for iPLEX reactions is listed in FIG. 3. The concentrations of the extension primers were further adjusted to compensate differences in signal intensities in mass spectra due to differences in sequence and molecular weight among different extension primers. Adjustments were made based on real mass spectral data generated. The concentration of each primer is listed in FIG. 6.

Step 4: MS Analysis

Extension products were desalted for MS analysis by adding 16 μL ddH₂O and 6 mg SpectroCLEAN resin. After centrifugation at 360 g for 5 min, approximately 15 mL of reaction solution was dispensed onto a 384-sample format SpectroCHIP using a Nanodispenser. Data acquisitions from SpectroCHIP were performed by Bruker Compact MALDI-TOF MS (Bruker Daltonics, Billerica, Mass., USA) and data analysis was carried out using TyperAnalyzer Application, version 4.0 (Sequenom, Inc.).

A representative mass spectrum for multiplex assay 1 is shown in FIG. 1.

Results

Upon optimizations of PCR and primer extension reactions, 88 treatment naive and 80 drug resistant serum samples were analyzed with a sensitivity close to 1,000 copies of HBV per milliliter of serum.

In this study, call rate was defined in two contexts: call rate per sample which was the successful call percentage for each individual sample among all variations; and call rate per variation which was the successful call percentage for each variation among all samples. When not specified, call rate referred to the successful call percentage for all variations among all samples.

To evaluate the performance of the HBV variation detection, we first looked at the call rate for each variation (defined as the percentage of successful calls in the selected samples tested for each variation). Thirty two variations (53.3%) achieved call rate over 98%. Fifty three variations (88.3%) achieved call rate over 90%. Fifty six out of the 60 variations (93.3%) achieved call rate over 80%. The overall call rate for all variations in all samples was 95.3%.

The call rates for each of the 60 variations in the two patient groups (treatment naive and drug resistant) are shown in FIG. 2 a. The overall call rates for all variations in treatment naive and drug resistant samples were 97.0% and 93.4%, respectively. Additionally, call rates of 14 variations in drug resistant patients were significantly lower than that in treatment naive patients.

Call rates per sample (defined as the percentage of the successful calls for all 60 variations tested in each sample) were also analyzed. In FIG. 2 b, call rates for each sample (its HBV viral load shown at X-axis) in the two patient groups were plotted. While call rates per sample remained high (>90%) over a wide range of concentrations for the treatment naive samples, a number of drug resistant samples had less than optimal call rates (7 with call rates 80-90% and 4 with call rates <80%).

Four of the 60 variations had call rates below 80%, which was the main reason for the overall no-call rate of 4.7%. None of these four variations has known functions. Additionally, there was a performance bias against drug resistant samples since all 11 samples with call rates below 90% were drug resistant samples.

Example 4 Capillary Sequencing Validation

A total of 70 samples (35 treatment naive and 35 drug resistant) were randomly selected for sequencing to validate MS results. The same PCR products used for SAP treatment (see Example 3) were used for direct sequencing using the BigDye Terminator Cycle Sequencing Kit, Version 3.0 (Applied Biosystems) and the AM 3100 Genetic analyzer (Applied Biosystems).

The sequencing primers were

(SEQ ID NO: 65) 5′-gttggatgGACTCGTGGTGGACTTCTCTCA-3′ (PCR primer), (SEQ ID NO: 66) 5′-ggatgGAGAGTAACTCCACAGTAGCTCCAA-3′ (PCR primer), (SEQ ID NO: 67) 5′-GYGCCATTTGTTCAGTGGTTCG-3′ and (SEQ ID NO: 68) 5′-AAACATAGAGGTTCCTTGAGCAGG-3′.

For the reverse transcriptase region, 27 treatment naive and 33 drug resistant samples were sequenced successfully. For the precore promoter and basal core promoter region, 29 treatment naive and 33 drug resistant samples were sequenced successfully. The overall sequencing success rate was 87%, although all the samples were successfully analyzed by MS.

Excluding some failed calls for certain variations in some samples, a total of 3,380 variation calls were obtained for both MS and direct sequencing. Overall, 3,340 variation calls (98.8%) were completely concordant between MS and sequencing, regardless of whether only a wild type or a mutant was present, or both sequences were present. For 23 variation calls (0.7%), MS detected both a wild type and a mutant while sequencing detected only a wild type or a mutant. One representative example is shown in FIG. 5 a. For these calls, it is possible that sequencing missed the minor sequence (either wild type or mutant) due to a lower frequency of the minor variation (<20%). Some of these cases were validated by further cloning and sequencing.

For 12 variation calls (0.4%), sequencing detected both a wild type and a mutant while MS detected only wild type or mutant. Majority of these MS calls were of lower signal quality, which is likely to cause inability to detect a minor variation (FIG. 5 b). For 0.1% of calls (5 out of 3,380 calls), direct sequencing and MS were completely discordant. Further cloning and sequencing confirmed that direct sequencing was correct. Highly consistent data were obtained between direct sequencing and MS, with only 5 variation calls out of 3,380 calls completely discordant. More frequently, MS detected the presence of both a wild type sequence and a mutation while direct sequencing did not. It should be noted that virtually all heterozygous calls from direct sequencing were done by careful manual inspection, while virtually all calls (homozygous or heterozygous) from MS were done automatically by the software. Additionally, sequencing reactions failed in 13% of the amplicons that were successfully analyzed by MS.

The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

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1. A method for the detection of hepatitis virus (HV) variations, comprising, a) providing a DNA molecule of an HV from a subject suffering from the HV infection as a template, wherein the DNA molecule includes an HV variation site; b) extending multiplex extension primers along the template to obtain extension products containing nucleotides at the variation sites; and c) analyzing the extension products by mass spectrometry (MS) to detect the HV variations.
 2. The method according to claim 1, wherein the HV is a Hepatitis B virus (HBV).
 3. The method according to claim 1, wherein the DNA molecule is provided by conducting a 2-plex PCR using the primers having a 70%, 80%, 85%, preferably 90%, more preferably 95%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 1-4.
 4. The method according to claim 1, wherein the template is provided after shrimp alkaline phosphatase (SAP) treatment.
 5. The method according to claim 1, wherein the multiplex extension primers are extended by conducting a 12-plex iPLEX reaction using the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 5-16, by conducting a 17-plea iPLEX reaction using the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 17-33, by conducting a 17-plex iPLEX reaction using the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 34-50, or by conducting a 14-plex iPLEX reaction using the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 51-64.
 6. The method according to claim 1, wherein the MS is matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).
 7. A method for the treatment of a hepatitis virus (HV) infection in a subject, comprising, a) detecting HV variations in the subject according to the method of claim 1, wherein the HV variations are in association with drug resistance; b) assessing the drug resistance of the subject based on the detection; and c) treating the subject based on the assessment.
 8. The method according to claim 7, wherein the HV is a Hepatitis B virus (HBV).
 9. The method according to claim 7, wherein the DNA molecule is provided by conducting a 2-plex PCR using the primers having a 70%, 80%, 85%, preferably 90%, more preferably 95%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 1-4.
 10. The method according to claim 7, wherein the template is provided after shrimp alkaline phosphatase (SAP) treatment.
 11. The method according to claim 7, wherein the multiplex extension primers are extended by conducting a 12-plex iPLEX reaction using the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 5-16, by conducting a 17-plex iPLEX reaction using the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 17-33, by conducting a 17-plex iPLEX reaction using the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 34-50, or by conducting a 14-plex iPLEX reaction using the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 51-64.
 12. The method according to claim 7, wherein the MS is matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).
 13. A kit for the detection of hepatitis virus (HV) variations by mass spectrometry analysis, comprising multiplex extension primers as described in claim
 1. 14. The kit according to claim 13, wherein the HV is a Hepatitis B virus (HBV).
 15. The kit according to claim 13, wherein the multiplex extension primers are the primers having a 70%, 80%, 85%, 90%, 95%, 96%, 97%, preferably 98%, more preferably 99%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 5-16, SEQ ID NOs: 17-33, SEQ ID NOs: 34-50 or SEQ ID NOs: 51-64.
 16. The kit according to claim 13, further comprising amplification primers, preferably the amplification primers having a 70%, 80%, 85%, preferably 90%, more preferably 95%, even more preferably 100% identity with those as set forth in SEQ ID NOs: 1-4.
 17. The kit according to claim 13, further comprising reagents for PCR.
 18. The kit according to claim 13, further comprising reagents for primer extension.
 19. The kit according to claim 13, further comprising reagents for dephosphorylation, preferably shrimp alkaline phosphatase (SAP).
 20. The kit according to claim 13, further comprising reagents as contained in MassARRAY iPLEX™. 